Anthracycline chemotherapeutics, such as doxorubicin (Doxo), are among the most effective and widely used antineoplastic drugs, yet their clinical application is limited by the damaging cardiac side effects that occur in many patients. Anthracycline-induced cardiotoxicity (AIC) can manifest acutely during cancer treatment, but can also cause life threatening cardiomyopathy and heart failure that develops years after the cessation of chemotherapy. Despite significant effort, the underlying mechanisms responsible for AIC are not fully defined, and it remains impossible to predict which patients will experience cardiotoxicity. Thus, there is an urgent need to advance mechanistic understanding in order to discover novel treatments and/or predictive biomarkers for this devastating condition. The overall objective of this proposal is to comprehensively define how type I interferon (IFNab) signaling, a pleiotropic innate immune pathway, potentiates the cardiotoxic effects of Doxo chemotherapy. The central hypothesis is that Doxo-induced cardiac DNA damage triggers the Stimulator of Interferon Genes (STING)-dependent production of IFNab, in turn driving a self-propagating cycle of mitochondrial dysfunction, reactive oxygen species (ROS) production, and cardiomyocyte death that contributes to cardiac remodeling and failure. In support of this hypothesis, ongoing studies have revealed that Doxo robustly engages STING to upregulate IFNab responses in cardiac cells and tissue. Strikingly, both male and female mice lacking STING or IFNab signaling are protected from Doxo-induced cardiac mitochondrial damage, myocardial remodeling, and left ventricle dysfunction. Conversely, treating melanoma-bearing mice with adjuvant IFNa in addition to Doxo results in enhanced cardiac fibrosis and contractility defects relative to tumor-bearing mice receiving Doxo alone. To gain additional insight into what appears to be an unappreciated yet fundamental driver of AIC, three related, but independent, aims are proposed. Aim 1 will employ single-cell approaches, a novel IFNab reporter mouse, and conditional knockout lines to define the kinetics of IFNab production in the heart and determine that STING-IFNab signaling in cardiac myeloid and myocyte populations contributes to AIC. Aim 2 will test the hypothesis that STING-IFNab signaling amplifies the cardiotoxicity of anthracyclines by potentiating mitochondrial dysfunction, iron overload, and oxidative stress. Finally, Aim 3 will establish that the IFNab-mediated upregulation of Z-DNA binding protein 1 (ZBP1) enhances sensing of cardiac DNA damage and promotes cardiac necroptosis to sustain inflammatory responses during and after Doxo chemotherapy. This proposal is innovative because it expands the current paradigms of AIC and defines the IFNab-mitochondrial nexus as a fundamental pathway contributing to myocardial damage. In addition, this research will contribute significant new information on how cardiac innate immune responses potentiate heart failure. In the long term, this work may positively impact human health by establishing IFNab as a predictive biomarker for cardiotoxicity and/or revealing novel therapeutic approaches to combat cancer therapy-related cardiac dysfunction.